1. Field of the Invention
The present invention relates to a labeled probe array, a method for producing the labeled probe array, and an method for determining the amount of a target substance using the labeled probe array.
2. Related Background Art
Recently, for precise assessment of a target substance, it has been intensively studied a method using a plurality of probes disposed in an array on the surface of a substrate, called “probe array”. A typical example is a so-called “DNA probe array” in which a plurality of DNA probes are disposed on a substrate. This DNA probe array is also referred to as a DNA chip. By using the DNA probe array, for example, the presence of a plurality of target genes can be analyzed at the level of base sequence.
Methods of making the probe array are roughly classified into two.
One is a method that synthesizes probes by sequential synthesis on a solid substrate, applicable to the probes being a substance that can be synthesized by chemical elongation and of which nucleotide or amino acid sequence has an important meaning, e.g., DNA, oligonucleotide, peptide nucleic acids (PNAs), proteins, and oligopeptides.
As an example of such a method, U.S. Pat. No. 5,445,966 discloses a method of synthesizing a DNA probe array on a substrate by using photolytic protection groups and photolithography. Alternatively, U.S. Pat. No. 5,474,796 discloses a method supplying nucleotide monomers by an ink jet process.
The other method is applicable, in addition to the above described substances, to the substances not chemically synthesizable, since this method is to supply probes that have been previously synthesized or prepared to the matrix sites of a probe-array substrate by the micro-dispensing method, pin transfer method, or ink jet process etc.
Since the second method uses probes which have been purified and of which quantity can be controlled, the purity of the probes are secured and the concentrations of the probes to be reacted with the substrate can be freely determined. Consequently, the final amounts of respective probes on the substrate surface can be assessed with a certain accuracy, to enhance the reliability of the target substance detection using the probe array. On the other hand, the second method requires previous synthesis of all kinds of probes for the probe array and it also requires to apply the probes to the substrate for reaction. Sometimes, several hundreds of thousands of probes may be required for a probe array. In that case, it will be considerably difficult to synthesize all probes beforehand.
On the other hand, according to the first method, necessary probes are synthesized on the substrate and then used for the reaction with the target substances, so that operations for immobilizing the previously synthesized or prepared probes onto the substrate can be omitted.
However, in order to improve the detection sensitivity or reliability of the first method, following points must be improved.
(1) The yield in each step of the sequential synthesis is not 100%, some times the final yield is as low as 5%. This directly leads to reduction in the sensitivity when the target substance is detected.
(2) For the same reason, both full length probes and shorter length probes are present at each matrix site of the probe array. This lowers the reliability in detecting a target substance.
(3) As a result of in situ synthesis on the substrate, the amount of the probe at each matrix site cannot be measured. This also lowers the reliability in detecting a target substance.
(4) As mentioned above, the synthetic yield in each step of sequential synthesis is not 100% and the synthetic yield varies depending on the steps and the matrix sites, so that the final amounts of respective probes vary broadly. This also lowers the reliability of the detection of a target substance.